PROJECT SUMMARY Lysine acetylation has traditionally been studied as an epigenetic modifier of histone tails within chromatin that provides an important mechanism for regulating gene expression. In the heart, histone acetylation acts as a key regulator of cardiac remodeling and function. However, recent reports have shown that non-histone proteins can be acetylated. Importantly, it has been postulated that the acetylome rivals phosphorylation in prevalence as a post-translational modification. The long-term goal of my lab is to dissect the epigenetic and non-epigenetic actions of lysine acetylation in the regulation of heart failure. The objective of this application is to elucidate the role of acetylation of non-histone proteins in the regulation of cardiac muscle function, with an emphasis on sarcomeric proteins. Mutations or modifications of sarcomeric proteins are known to drive cardiac pathology that results in heart failure. Phosphorylation of sarcomeric protein function has been widely studied, yet very little is known about the impact for other post-translational modifications in the regulation of muscle protein function. Preliminary findings from our lab using mass spectrometry demonstrated that obesity-mediated cardiac remodeling is associated with significant changes in lysine acetylation of proteins within the left ventricle of mice. Ingenuity Pathway Analysis identified the Cardiovascular Disease Network and revealed LIM domain-binding protein 3 (LDB3) and skeletal muscle alpha actin 1 (ACTA1) as proteins that were significantly impacted by obesity. LDB3 and ACTA1 affect muscle structure, integrity, and cellular motility; mutations in these two proteins contribute to cardiomyopathy. Thus, post-translational modification of these proteins likely elicits functional changes that would result in muscle cell dysfunction. Whether acetylation or deacetylation of these proteins impacts heart function remains unknown. However, preliminary data suggests that (de)acetylation of ACTA1 impacts actin-myosin interaction and kinetics. This led to the central hypothesis that sarcomere protein (de)acetylation, specifically LDB3 and ACTA1, regulates cardiac muscle cell function. Three specific aims were developed to test this hypothesis. (Aim 1) Delineate a role for ACTA1 protein (de)acetylation in cardiac muscle contractility and function. (Aim 2) Elucidate the role for LDB3 acetylation in calcineurin-NFAT signaling and pathological cardiac hypertrophy. (Aim 3) Use CRISPR/Cas technologies to determine the physiological significance of LDB3 and/or ACTA1 (de)acetylation in vivo. Most studies to date have examined lysine acetylation in the regulation of nucleosomal DNA and gene expression. In addition, most studies examining sarcomere protein modifications have focused on phosphorylation. As such, the proposed research is innovative and will add significant insight into the process of sarcomere protein (non-histone) (de)acetylation in the regulation of cardiac biology.